4668
a
J.-P. Wu et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4664–4669
quenching with excess DTT and purification by Sephadex
b
G-25 gel filtration. A mouse monoclonal anti-phospho-
LSP1 IgG1 antibody was generated to mcKLH (Pierce)
conjugated phospho-peptide antigen, CRTPKLARQA-
pS-IELPSM, from AnaSpec. The anti-phospho LSP1
IgG1 antibody was labeled with the Eu-chelate of N1-(p-
isothiocyanatobenzyl)-diethylenetriamine-N1,N2,N3,N3-tet-
raacetic acid (Perkin Elmer Life Sciences) for the DELFIA
of MK2 catalysis and inhibition. Compounds and acti-
vated GST-MK2a 1-400 (1 nM) were incubated in neutra-
vidin coated plates (Pierce) at 25 °C in the following 40 lL
reaction mixture for 30 min: 50 mM HEPES (pH 7.6),
50 mM KCl, 10 mM MgCl2, 100 lM Na3VO4, 0.01%
CHAPS, 1 mM DTT, 10 lg/mL bovine serum albumin, 1%
DMSO, 500 nM biotinylated GST-LSP1 179–339, and
2 lM ATP. The plates were washed with 25 mM Tris–HCl
(pH 7.5), 150 mM NaCl, and 0.05% Tween 20. Eu-chelated
anti-phospho-LSP1 IgG1 antibody (0.5 mg/mL) was
diluted 1:20,000 in 50 mM Tris–HCl (pH 7.5), 150 mM
NaCl, 10 lM DTPA, 0.05% Tween 40, 0.2% bovine serum
albumin, and 0.05% BGG; 40 lL of the diluted antibody
solution was added to the plates. After 1-h incubation at
25 °C, the plate was washed prior to the addition of
DELFIA enhancement solution (Wallac #4001-0010), and
read after 15 min at exk = 360 nm and emk = 620 nm.
8. The synthesis of the C4-monomethyl analog 17 is
considerably easier than that of C4-gem-dimethyl analog
3. Since compounds 17 and 3 are comparable in potency,
we chose compound 17 as the reference compound for
initial SAR studies. Later studies (to be published in
separate papers) showed that in more advanced struc-
tures, the C4-gem-dimethyl moiety improved potency
moderately.
M138
76
H108
K93
E104
D207
P-loop
(red)
L141
2.6 Å
D142
L70
3.2 Å
N191
E145
Figure 3. X-ray structure of MK2a [41–364] co-crystallized with
compound 76.
Supplementary data
Experimental procedures for crystal complex formation
and X-ray data collection; synthetic schemes for com-
pounds in Figure 2. This material is available free of
charge via internet. Supplementary data associated with
this article can be found, in the online version, at
References and notes
1. Robinson, M. J.; Cobb, M. H. Curr. Opin. Cell Biol. 1997,
9, 180.
2. There are four major families of MAPKs: (1) the
archetypal extracellular regulated kinases (ERKs), (2)
the c-jun N-terminal kinases (JNKs), (3) the p38 MAPKs,
and (4) the ERK5 or BigMAPKs, see: Tibbles, L. A.;
Woodgett, J. R. Cell. Mol. Life Sci. 1999, 55, 1230.
3. (a) Jackson, P. F.; Bullington, J. L. Curr. Topics Med.
Chem 2002, 2, 1009; (b) Cirillo, P. F.; Pargellis, C. A.;
Regan, J. Curr. Top. Med. Chem. 2002, 2, 1021, and
references cited therein.
9. (a) The X-ray coordinates have been deposited with RCSB
Protein Data Bank, deposition #2PZY; The protein used
in this study is a segment of MK2a containing residues 41-
364. For a detailed crystal structure of a different MK2
complex, in which MK2 is bound to its activator p38, see:
(b) White, A.; Pargellis, C. A.; Studts, J. M.; Werneburg,
B. G.; Farmer, B. T., II Proc. Natl. Acad. Sci. U.S.A.
2007, 104, 6353.
10. For the THP-1 assay protocol, see: Regan, J.; Breitfelder,
S.; Cirillo, P.; Gilmore, T.; Graham, A. G.; Hickey, E.;
Klaus, B.; Madwed, J.; Moriak, M.; Moss, N.; Pargellis,
C.; Pav, S.; Proto, A.; Swinamer, A.; Tong, L.; Torcellini,
C. J. Med. Chem. 2002, 45, 2994.
4. Kotlyarov, A.; Yannoni, Y.; Fritz, S.; Laaß, K.; Telliez,
J.-B.; Pitman, D.; Lin, L.-L.; Gaestel, M. Mol. Cell. Biol.
2002, 22, 4827.
5. Dinarello, C. A. Nutrition 1995, 11, 492; . Rev. Infect. Dis.
1984, 6, 51; Curr. Opin. Pharmacol. 2004, 4, 378.
6. (a) A selection of patent and patent applications claiming
MK2 inhibitors is listed below Meyers, M. et al.
WO2005009370, US20050143371, US20050137220, US
20050101623; (b) Anderson, D. R. et al. WO2004055015;
WO2004054505; WO2004054504; US2004127519, US2004
127511, US2004 142978; (c) Vazquez, M. L. et al. US20
04127492; (d) Buzon, R. A. et al. WO2004020440; WO
2004020438; (e) Sato, H. et al. WO2005007092; (f)
Kataoka, K. et al. WO2004076458; (g) Aronov, A. et al.
WO2004037814; (h) Hodge, C. N. et al. WO2005/033102;
(i) Wyatt, P. et al. WO2005014554
7. A dissociation-enhanced lanthanide fluorescent immuno-
assay (DELFIA) of MK2 catalysis was employed to
determine the potency of MK2 inhibition, see: (a) Lukas,
S. M.; Kroe, R.; Wildeson, J.; Peet, G. W.; Frego, L.;
Davidson, W.; Ingraham, R. H.; Pargellis, C. A.; Labadia,
M. E.; Werneburg, B. G. Biochemistry 2004, 43, 9950; (b)
Assay protocol: The GST-MK2a 1–400 variant of MK2
was activated with p38a MAPK by methods similar to
those described previously7 and re-purified by GSH
affinity chromatography. The GST-LSP1 179–339 sub-
strate of MK2 was biotinylated with a 70-fold molar
excess of PEO-iodoacetyl biotin (Pierce), followed by
11. It is conceivable that poor membrane permeability
contributed to the lack of cellular potency for compounds
with terminal polar groups. This was investigated subse-
quently, the results of which will be published separately.
12. The selectivity against MK1a and ERK2 was determined
in an IMAP assay, see: (a) Sportsman, J.; Daijo, J.;
Gaudet, E. A. Comb. Chem. High Throughput Screening
2003, 6, 195; (b) Assay protocol: Compounds were diluted
with DMSO in kinase assay buffer (10 mM Tris–HCl (pH
7.2), 10 mM MgCl2, 0.01% Tween 20, and 1 mM DTT)
such that the final concentration of DMSO was 1%. The
diluted compounds were incubated with kinase, ATP and
peptide substrate in Corning 96-well half-area, black non-
binding surface microtiter plates in kinase assay buffer for
90 min at room temperature. The MK1a reaction con-
sisted of 250 pM MK1a (Upstate Catalog #14-509), 1 lM
ATP, and 100 nM FAM-S6 ribosomal protein-derived
peptide. The ERK2 reaction consisted of 100 pM ERK2
(Upstate Catalog #14-173), 2.5 lM ATP, and 100 nM
FAM-Erktide. The kinase reactions were halted by
addition of IMAP progressive binding reagent in each
well. After 30 min of incubation at room temperature, the
plates were read for fluorescence polarization on an